Inflammation is a defense mechanism of the body against the harmful stimuli. In inflammation, the immune system removes the harmful stimuli and begin the healing process by involving immune cells, blood vessels, and molecular mediators. It is one of the significant and fraught processes in human body. But sometimes immune system triggers an inflammatory response which induces overproduction of coagulant protein known as thrombin causing dangerous blood clots and other condition.
A naturally occurring anticoagulant protein -Activated protein C (APC) having the anti-thrombotic property is used to treat severe blood infections and wounds by reducing inflammation of the endothelial cells that line blood vessels. The major disadvantage of APC is that it can affect blood clotting ability by inhibiting thrombin too much which in turn increases bleeding risk.
Now, a collaborative team of researchers from the Division of Hemostasis and Thrombosis at Beth Israel Deaconess Medical Center (BIDMC) and the Wyss Institute at Harvard University have found a new semi-synthetic molecule called “parmodulins” which mimics like APC but do not interferes with normal blood clotting and coagulation making it a new attractive drug candidate. This work was enabled by leveraging the Wyss Institute’s Organ-on-a-Chip technology to model thrombosis within a human blood vessel in vitro. The results are reported in this week’s issue of Proceedings of the National Academy of Sciences.
According to former Wyss postdoc Abhishek Jain, Ph.D., who is now an Assistant Professor and director of the Bioinspired Translational Microsystems lab at Texas A&M University, the team did a mini pre-clinical trial to see the effect of parmodulins on the endothelium. During this trial, they found the pathway of parmodulin function as well as revealed the mechanism of action of parmodulin i.e. how they protect endothelial cells from damage.
The target protein PAR1 which is a receptor for thrombin on which both APC and parmodulins act, is present on both endothelial cells and platelets that circulate through the blood and promote clotting, making mechanistic analysis difficult. However, when PAR1 is activated by APC on endothelium, it triggers anti-inflammatory, anti-apoptotic, and barrier-fortifying pathways, all of which help protect cells from the negative effects of inflammation.
In addition to activating PAR1, APC also inhibits the generation of the essential component of blood clotting i.e thrombin, but inhibiting thrombin too much leads to uncontrolled bleeding. In the light of the knowledge that parmodulins bind to PAR1, the team wanted to find a manner which not only activates endothelial PAR1 but reduce thrombic responses without thinning the blood, and thus provide a better alternative to APC.
In the laboratory, Karen De Ceunynck, Ph.D. incubated human endothelial cells with parmodulin 2 in vitro for 4 hours. After incubation, it was exposed to lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α) ( thrombin-inducing inflammatory agents). They observed that in parmodulin-exposed cells, the thrombin generating ability was reduced by 50% as compared with non-parmodulin-exposed cells. In spite this, parmodulin 2 did not inhibit the activity of factor V or factor X, proteins that function in healthy blood coagulation.
“We were compelled by the idea that parmodulin 2 inhibited LPS- and TNF-mediated prothrombotic effects on the endothelial surface without impairing blood clotting,”
The team used a Wyss-developed blood-vessel-on-a-chip to evaluate the response of the endothelium for confirmation. Blood-vessel-on-a-chip consists of microfluidic channels embedded in a clear polymer chip, coated with collagen, and lined by human endothelial cells. In order to simulate the flow conditions within human blood vessels, whole blood was perfused through the chip, and added different pro- and anti-inflammatory compounds to evaluate the response of the endothelium.
Researchers observed that platelets accumulated on the endothelium in a typical inflammatory response on exposure to TNF-α. But if it was exposed to parmodulin 2 before TNF alpha, endothelium resumed its normal function because the accumulation of platelets did not take place. Scientists concluded that thrombotic response of a endothelium is blocked on exposure to parmodulin without affecting blood coagulation in humans – a noteworthy advance over APC.
We observed that the cytoprotective response induced by parmodulin 2 happened very quickly, and confirmed its rapid onset in time course and gene expression assays.
In the laboratory, they found that in mice binding of WBC to blood vessels is reduced by parmodulin 2 and inhibited the inflammatory response by blocking accumulation platelet and fibrin. This finding confirms the anti-thrombotic and anticoagulant activity of parmodulin 2 in vitro. Additionally, they also observed that parmodulins do not interact with many of APC’s other binding partners, which makes it more targeted to PAR1 and reducing other side effects.
“The discovery of an anti-inflammatory molecule that prevents endothelial thrombosis but also preserves normal blood coagulation is a major step toward an alternative and better approach to treating inflammatory disease,” says Rob Flaumenhaft, M.D., Ph.D., Professor of Medicine at Harvard Medical School, Chief of the Division of Hemostasis and Thrombosis at BIDMC, and corresponding author of the paper. “Furthermore, nearly all other pharmaceuticals that target transmembrane PAR1-like receptors bind to the exterior side of the receptor; parmodulin 2 represents a paradigm shift for compounds targeting these receptors because it acts on the cellular side of the protein. We are excited to see if we can advance it to clinical trials.”
“This work provides another example of how organ-on-a-chip technology can enable faster and safer development and evaluation of drugs that could help patients around the world,” says co-author and Wyss Institute Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at HMS and the Vascular Biology Program at Boston Children’s Hospital, as well as Professor of Bioengineering at Harvard’s John A. Paulson School of Engineering and Applied Sciences (SEAS).
This research was supported by the National Heart, Lung, and Blood Institute and the Wyss Institute for Biologically Inspired Engineering at Harvard University.
Source: Materials Provided by Wyss Institute at Harvard University
Note: Content edited for style and length
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